PROJECT 4 Neuroactive steroids are effective modulators of gamma-aminobutyric acid-A receptors (GABARs), augmenting the actions of GABA at low concentrations and directly activating GABAR chloride channels in the absence of GABA at higher concentrations. Other steroids, particularly those with charged substituents at the CS-position, are negative modulators of GABARs and either potentiators or inhibitors of N-methyl-D- aspartate receptors (NMDARs). Present evidence indicates that GABAR potentiation and NMDAR inhibition represent two key mechanisms in clinical anesthesia. Over the several years, we have developed tools that have helped us to probe the effects of neuroactive steroids on GABARs, including a series of novel steroid enantiomers and fluorescent steroids that have provided important new information about intracellular and intramembranous accumulation of these agents. One of the fluorescent steroids only potentiates GABARs when exposed to visible (-480 nm) light, offering the potential to develop a new class of GABAergic modulators that can be activated in specific regions of cells or brain. In the present proposal, we will extend our studies of neuroactive steroids by addressing four goals: 1. To examine membrane and receptor mechanisms contributing to the effects of GABA potentiating 3-alpha-hydroxysteroids; 2. To examine membrane mechanisms contributing to the effects of C3-sulfated steroids and 3-beta-hydroxysteroids on GABARs; 3. To examine mechanisms contributing to the effects of photoactive steroids on GABARs; and 4. To gain new information about structural requirements for steroids on GABARs and NMDARs. These studies will use a combination of physiological and cellular imaging methods to examine steroid effects in cultured rat hippocampal neurons, and HEK cells and Xenopus oocytes expressing defined GABAR subunits, and will involve a combination of structure-activity and mechanistic studies based upon our initial observations with the novel fluorescent and photoactive steroid analogues. These studies have the potential to provide new information about neuroactive steroid effects in the CMS and a better understanding of mechanisms involved in steroid-mediated anesthesia.